Multiplex Detection Kit

Multiplex IHC Detection Kit (Triple)

Cat. Num. AAA266514

• Applications: Immunofluorescence, Immunohistochemistry
• Synonyms: Multiplex; N/A; Multiplex IHC Detection Kit (Triple); Multiplex detection kit

• Applicable Applications for Multiplex detection kit: IF (Immunofluorescence), IHC (Immunohistochemistry)
• Directions for Use: 1. De-paraffinizing (de-waxing) and rehydrating; 1.1 Heat the slides in tissue-drying oven for 45 minutes at 60 degree C. Place the slides in a rack, and perform the following washes: Xylene I: 15 mins Xylene II: 15 mins 100% Ethanol: 5 mins 95% Ethanol: 5 mins 85% Ethanol: 5 mins 70% Ethanol: 5 mins; 1.2 The slides are placed in a lab draught cupboard to rinse off ethanol.; 1.3 Finally, wash the slides in the pure water.; 2. Antigen or epitope retrieval; 2.1 Add the appropriate antigen retrieval buffer (EDTA pH 9.0 or sodium citrate pH 6.0) to the microwaveable vessel.; 2.2 Place the slides in the microwaveable vessel. Then place the vessel inside the microwave (1200W).; 2.3 Boil for 8 mins in microwave (1200 W) under medium heat, then stop heating for 8 mins, followed by low and medium heat for 7 mins. Other heat-induced epitope retrieval methods can also be used, e.g., heated at 120 degree C 1-2 min, 100 degree C 20mins or 95 degree C in a water bath. Be sure to monitor for evaporation and watch out for boiling over during the procedure. Do not let the slides dry out.; 2.4 After cooling down in room temperature, place the slides in PBS (pH 7.4) to wash 3 X 5 mins on a decolorizing shaker.; Notes: To get best results, antigen retrieval buffer and protocol should be determined according to the tissue types and antigen types.; 3. Blocking endogenous peroxidase; 3.1 Add enough 3% hydrogen peroxide (H2O2) to cover the slides.; 3.2 Incubate for 15 mins in the dark at room temperature.; 3.3 Place the slides in PBS (pH 7.4) to wash 3 X 5 mins on a decolorizing shaker.; 4. Blocking; 4.1 Drain slides and then use an IHC pen to draw a circle around each sample on your slide (to hold antibody solution within the target area).; 4.2 Add 3% BSA-PBST solution (or other blocking buffer) inside the circle to cover the tissues, incubate 30 mins at room temperature.; 5. Primary antibody incubation; 5.1 Remove blocking buffer and add primary antibody diluted by recommended antibody diluent overnight at 4 degree C or 37 degree C for 1-2h.; 6. HRP Polymer Working Solution incubation; 6.1 Place the slides in PBS (pH 7.4) and wash 3 X 5 mins on a decolorizing shaker.; 6.2 Incubate slides with HRP Polymer Working Solution (50ul for each slice) in the dark at room temperature for 60 mins.; 6.3 Wash 3 X 5 mins with PBS buffer.; 7. Tyramide labeling; 7.1 Apply the TSA-520 Dye Working Solution to each sample (50ul for each slice) and incubate for 10-15 mins at room temperature.; 7.2 Wash 3 X 5 mins with PBS buffer.; 8. Denoise Repeat steps 2. At the end of the single stain, may add antifade mounting medium to view the slides or go on labeling another fluorescent dye.9. RepeatRepeat steps 3-7 for another fluorescent dye (TSA-620 Dye Working Solution) to each sample.; 10. DAPI counterstaining; 10.1 Place the slides in PBS (pH 7.4) and wash 3 X 5 mins on a decolorizing shaker.; 10.2 Apply the DAPI solution (50ul for each slice) to each sample and incubate in the dark at room temperature for 10 mins.; 11. Mounting the slides; 11.1 Place the slides in PBS (pH 7.4) and wash 3 X 5 mins on a decolorizing shaker.; 11.2 Add antifade mounting medium (3-5ul for each slice) to cover the section.; 12. View the slides. View the sample using a fluorescence microscope with appropriate filters.; Note: To get the best results from multiplex staining, the experimental condition must be optimized.
• Preparation and Storage: Store at 4 degree C in dark for 1 year, do not freeze.

Application Data

Related Product Information for Multiplex detection kit

Three colors multiplex IHC tyramide signal amplification (TSA)